Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

• Published in: Biochemistry (Easton) Vol. 28; no. 8; pp. 3145 - 3152
• Main Authors: Koch, John A, Waxman, David J
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 18.04.1989
• Subjects: 550201 - Biochemistry- Tracer Techniques; AMINO ACIDS; AMP; ANIMAL CELLS; ANIMALS; BASIC BIOLOGICAL SCIENCES; BETA DECAY RADIOISOTOPES; BETA-MINUS DECAY RADIOISOTOPES; Binding Sites; BIOCHEMISTRY; CARBOXYLIC ACIDS; CELL CONSTITUENTS; CHEMICAL REACTIONS; CHEMISTRY; Cytochrome P-450 Enzyme System - metabolism; CYTOCHROMES; DAYS LIVING RADIOISOTOPES; ELECTROPHORESIS; ENZYME ACTIVITY; Enzyme Induction - drug effects; ENZYMES; GLUCAGON; HORMONES; HYDROXY ACIDS; In Vitro Techniques; IN VIVO; ISOTOPES; LIGHT NUCLEI; Liver - drug effects; Liver - enzymology; LIVER CELLS; Male; MAMMALS; MICROSOMES; NUCLEI; NUCLEOTIDES; ODD-ODD NUCLEI; ORGANIC ACIDS; ORGANIC COMPOUNDS; ORGANOIDS; Oxygenases - metabolism; PEPTIDE HORMONES; PEPTIDES; Phenobarbital - pharmacology; PHOSPHORUS 32; PHOSPHORUS ISOTOPES; PHOSPHORUS-GROUP TRANSFERASES; PHOSPHORYLATION; PHOSPHOTRANSFERASES; PIGMENTS; POLYPEPTIDES; POST-TRANSLATION MODIFICATION; Protein Kinases - metabolism; Protein Processing, Post-Translational; PROTEINS; RADIOISOTOPES; RATS; Rats, Inbred F344; RIBOSOMES; RODENTS; SERINE; SOMATIC CELLS; TRANSFERASES; VERTEBRATES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00434a005

Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [32P]orthophosphate in the presence of N6,O2'-dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-inducible P-450 form PB-4 (P-450 gene IIB1). Little or no phosphorylation of these proteins was observed in the absence of diBtcAMP or glucagon or in the presence of activators of Ca2+-dependent protein kinases. Two-dimensional gel electrophoresis revealed that these 32P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 microM. In contrast, little or no phosphorylation of P-450 forms reactive with antibodies to P-450 PB-1 (gene IIC6), P-450 2c (gene IIC11), or P-450 PB-2a (gene IIIA1) was detected in the isolated hepatocytes under these incubation conditions. Phosphoamino acid analysis of the 32P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2.